Transcriptional Control of Cyclin D1 · Transcriptional Control of Cyclin D1 Ying Zhang1,2*, ... the total protein lysate was analyzed by western blotting using an anti-FLAG monoclonal

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EF Hand Protein IBA2 Promotes Cell Proliferation in Breast Cancers via

Transcriptional Control of Cyclin D1

Ying Zhang1,2*, Shuling Wang2 and Lingsong Li2,3*

1State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese

Academy of Sciences, Beijing 100101, China

2Department of Cell Biology, School of Basic Medical Sciences, Peking University Health

Science Center, Beijing 100191, China; Peking University Stem Cell Research Center, China

National Center for International Research, Peking University Health Science Center, Beijing

100191, China;

3SARI Center for Stem Cell and Nanomedicine, Shanghai Advanced Research Institute,

University of Chinese Academy of Sciences, Shanghai 200120, China

*Co-corresponding author

Contact

Address correspondence to: Ying Zhang, E-mail: [email protected] and Lingsong Li,

E-mail: [email protected].

RUNNING TITLE: IBA2 in Breast Carcinogenesis

Disclosure of Potential Conflicts of Interest

The authors disclose no potential conflicts of interest.

Financial Support

This work was supported by the National Natural Science Foundation of China (grant no.

30900856 to Y. Zhang.) and China Postdoctoral Science Foundation (20080440010 and

200902024 to Y. Zhang.)

on June 23, 2020. © 2016 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 4, 2016; DOI: 10.1158/0008-5472.CAN-15-2927

http://cancerres.aacrjournals.org/

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Abstract

EF hand (EFh) domain-containing proteins have been implicated in malignant

progression but their precise functional contributions are uncertain. Here we report

evidence that the EFh protein IBA2 promotes the proliferation of breast cancer cells by

facilitating their transit through the G1/S cell cycle transition. Mechanistic

investigations revealed that IBA2 acted at the transcriptional level to promote the

expression of the critical cell cycle regulator cyclin D1. Clinically, we found that levels of

IBA2 were significantly upregulated in breast cancer specimens where its expression

correlated positively with histological grade. Our results identify suggest a key role for

IBA2 to mammary tumorigenesis.




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Introduction

EF hand (EFh) domain-containing proteins are involved in various disease states

including chronic inflammation, tumor progression, cardiomyopathy, and other clinical

disorders. They are associated with multiple targets that promote cell growth and

differentiation, cell cycle regulation and transcription activities (1,2). EFh proteins have

remarkable sequence homology and structural similarity, yet they function in a wide range of

biological processes. The activities of these proteins are tailored in part by a distinct pattern of

subcellular localization and tissue specific expression (3).

Ionized calcium-binding adapter molecule 1 (IBA1) is an EFh containing protein that

binds and crosslinks filamentous actin (F-actin) and localizes to membrane ruffles and

phagocytic cups. Daintain (also known as IBA1 or AIF-1) is closely associated with cardiac

allograft vasculopathy (4-6), rheumatoid arthritis (7), inflammatory skin disorders (8),

systemic sclerosis (9,10) and central nervous system injury (11-13). Furthermore, IBA1 plays

important roles in the initiation and progression of cancers, including breast cancer (14-19).

Ionized calcium-binding adapter molecule 2 (IBA2) is a homolog of IBA1 (20,21) and

share similar overall structure and molecular function with IBA1 (22). However, the distinct

expression patterns of the two proteins in various tissues of the body indicate different

functions between IBA1 and IBA2 (23). To date, the potential roles in carcinogenesis and the

cellular mechanisms of IBA2 have not been well characterized.

In this study, we demonstrate that IBA2 promotes the proliferation of breast cancer cells

by facilitating the G1/S transition through upregulation of cyclin D1. We found that IBA2 was




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frequently overexpressed in breast carcinomas, and that IBA2 expression levels were

positively correlated with tumor grades in breast carcinoma samples.

Materials and Methods

Bioinformatics

The mRNA length, open reading frame, conserved domains, and chromosome location of

IBA2 were predicted using NCBI databases (www.ncbi.nlm.nih.gov). The theoretical

molecular weight and isoelectric point of IBA2 were predicted using ExPASy

(www.expasy.ch/tools). Sequence alignments were performed with ClustalW (version 1.60)

(24), and phylogenetic analysis was performed using the Jotun Hein method (25).

Tissue specimens and cell lines

Breast carcinoma tissues from human patients were obtained from Beijing 301 Military

General Hospital (Beijing, China). Samples were frozen in liquid nitrogen immediately after

surgical removal and maintained at -80°C until use. All human tissues were collected

following protocols approved by the Ethics Committee of the Peking University Health

Science Center. Normal human breast epithelia cell and human breast cancer cell lines were

obtained from American Type Culture Collection (Manassas, VA).

Chromatin immunoprecipitation (ChIP) assays

ChIP assays were performed essentially as previously described (26). The following

primers were used to amplify the cyclin D1 promoter: forward: 5ʹ-tgccgggctttgatcttt-3ʹ and

reverse: 5ʹ-cggtcgttgaggaggttgg-3ʹ. The following primers were used to amplify a negative

control sequence: forward: 5ʹ-ttttggattttctttc-3ʹ and reverse: 5ʹ-gcattttgattttatt-3ʹ.

Tumor xenografts




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Tumor xenografts were performed as described previously (27). MCF-7 breast cancer

cells were plated and infected in vitro with mock (control) lentivirus encoding IBA2, or

lentivirus encoding IBA2 shRNA. After 48 h, 5 × 106 viable MCF-7 cells (in 200 mL PBS)

were injected into the mammary fat pads of 6 to 8 week old female BALB/c mice (Charles

River, Beijing, China). Six animals per group were used in each experiment. Seventeen

β-estradiol pellets (0.72 mg/pellet, 60 day release; Innovative Research of America, Sarasota,

FL) were implanted 1 day before the injection of MCF-7 cells. Tumor volume and weight

were measured. All studies were approved by the Animal Care Committee of Peking

University Health Science Center (Beijing, China).

Results

Cloning and characterization of IBA2

We cloned the IBA2 gene in GenBank (ID NM_031426) from a mammary cDNA library.

The cDNA of IBA2 is 3,455 bp in length and contains an open reading frame of 453 bp,

which includes one potential phosphorylation site and one potential acetylation site (Fig. 1A,

B). The predicted molecular mass and isoelectric point of IBA2 are ~17.0 kDa and 6.63,

respectively. The IBA2 gene is located on chromosome 9q34.13 and consists of five exons

and four introns. IBA2 is predicted to have an EFh domain, thus belonging to the EFh

proteins (Fig. 1A). These proteins have remarkable sequence homology and structural

similarity, yet they function in a wide range of biological processes. Based on protein

sequence, human IBA2 shares 82% identity with the mouse (Mus musculus) and rat (Rattus

norvegicus) homologs, 67% with the zebrafish (Danio rerio) homolog, and 75% with the




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chicken (Gallus gallus) homolog (Fig. 1B). Phylogenetic analysis also indicated that IBA2 is

an evolutionarily well-conserved gene (Fig. 1C).

To determine the expression profile of IBA2, we analyzed the expression of IBA2

mRNA in several mouse tissues (Fig. 2A). IBA2 mRNA expression was highest in the kidney,

relatively high in the heart, and low in the brain, spleen, lung, skeletal muscle and testis. IBA2

mRNA was not detected in the liver (Fig. 2A). We first examined the expression of IBA2

protein by transfecting a FLAG-tagged IBA2 expression construct into MCF-7 cells. After 48

h, the total protein lysate was analyzed by western blotting using an anti-FLAG monoclonal

antibody. As shown in Fig. 2B (left panel), IBA2 was expressed as a ~17 kDa protein,

confirming its predicted molecular weight. We also generated polyclonal antibodies to IBA2

using recombinant IBA2. Western blotting analysis of endogenous and overexpressed IBA2

protein using the anti-IBA2 polyclonal antibody confirmed that endogenous IBA2 is also ~17

kDa (Fig. 2B, right panel). In addition, IBA2 mRNA and protein expression are higher in

breast cancer cells than in normal human breast epithelial cells (MCF-10A) (Fig. 2C). To gain

insight into the biological function of IBA2, we examined its subcellular localization.

Overexpressed EGFP-IBA2 and FLAG-IBA2 localized to the cytoplasm and nucleus in HeLa

cells (Fig. 2D).

IBA2 promotes proliferation and tumorigenesis of breast cancer cells

Because of the crucial role of IBA1 in cell proliferation and tumorigenesis and its close

homology to IBA2, we hypothesized that the mis-regulation of IBA2 expression in breast

carcinomas and breast cancer cell lines may have pathological relevance. Thus, we

investigated whether IBA2 expression influenced breast cancer cell proliferation and




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tumorigenesis. First, we examined the effect of IBA2 overexpression or knockdown on cell

cycle regulation in mammary carcinoma cells. In these experiments, MCF-7 cells were

synchronized at the G0/G1 phase by serum starvation (28-30). Cell cycle profiling by FACS

indicated that IBA2 overexpression was associated with an increase in the transition of cells

to the S+G2/M phases from the G0/G1 phase (Fig. 3A). Consistently, knockdown of IBA2

with specific siRNA resulted in an accumulation of cells in the G0/G1 phase (Fig. 3B).

Collectively, these results indicated that IBA2 promotes the proliferation of breast cancer cells

by facilitating the G1/S transition.

To investigate the role of IBA2 in breast tumorigenesis, we overexpressed and knocked

down IBA2 by infecting MCF-7 cells using lentiviruses encoding the IBA2 gene or shRNA

against IBA2, respectively. A mock lentivirus (carrying an empty vector) and a lentivirus

encoding a non-silencing control shRNA were used as controls for overexpression and

knockdown, respectively. Colony formation assays demonstrated that overexpressing IBA2

increased the number of colonies formed, whereas knocking down IBA2 reduced the number

of colonies formed (Fig. 3C). To further demonstrate the importance of IBA2 in cell

proliferation, we performed BrdU incorporation assays, the indication of cell proliferation by

which is more sensitive. The number of BrdU-positive MCF-7 and human embryonic kidney

293 (HEK 293) cells increased when IBA2 was overexpressed and decreased when IBA2 was

downregulated (Fig. 3D, E). To demonstrate whether or not the oncogenic properties of IBA2

may also include an ability to promote migration, IBA2 overexpression and knockdown cells

were used in cell invasion assays. We found that neither IBA2 overexpression nor knockdown

affect cell invasion (Fig. S1A, B). Western blotting also showed the epithelial markers




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E-cadherin and α-catenin and the mesenchymal markers vimentin and S100A are expressed to

similar extents in both IBA2 overexpression and knockdown cells (Fig. S1C). These results

suggest that the oncogenic properties of IBA2 reside in its ability to promote proliferation

rather than migration.

To assess the importance of IBA2 on breast tumorigenesis in vivo, equal numbers of

IBA2-overexpressing and IBA2 shRNA-expressing MCF-7 cells and their respective controls

were implanted into the mammary fat pads of athymic BALB/c mice, and the growth of the

implanted tumors (n = 6 for each group) was measured over a period of 8 weeks.

Overexpression of IBA2 was associated with significant tumor growth, and knockdown of

IBA2 expression resulted in a dramatic reduction in tumor volume and weight (Fig. 4A, B,

C).

Identification of cyclin D1 as a downstream target for IBA2

To further dissect the signaling pathway underlying IBA2-mediated cell proliferation and

tumorigenesis, the expression of cell cycle-regulated genes was monitored by western blotting

and real-time PCR (Fig. 5A, B). As shown in Fig. 5A, whereas the overexpression or

knockdown of IBA2 had little effect on the protein expression of some cell cycle-regulated

genes, including cyclin E1, cyclin D3, cyclin B1, CDK2, CDK4, CDK6, and p-RB, the protein

and RNA expression of cyclin D1 was elevated in cells overexpressing IBA2 and reduced in

cells when IBA2 was downregulated. We also detected the senescence markers P53 and P21

and found that the overexpression or knockdown of IBA2 had little effect on the expression

level of both proteins (Fig. 5A). These results suggest that the cyclin D1 gene is a downstream

target of IBA2 in breast cancer cells.




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We further tested the role of cyclin D1 in promoting IBA2-dependent proliferation of

breast cancer cells by overexpressing cyclin D1 when IBA2 was knocked down to determine

whether cyclin D1 could alleviate the effect of G0/G1 accumulation induced by IBA2

knockdown. MCF-7 cells were co-transfected with either IBA2 siRNA and a cyclin D1

expression construct, or IBA2 siRNA and an empty expression vector as a negative control.

FACS analysis revealed that cyclin D1 expression in IBA2-downregulated cells resulted in a

significant decrease in the percentage of cells in the G0/G1 phase (Fig. 5C). These results

strongly suggest that cyclin D1 is a critical downstream mediator of IBA2 in promoting the

G1/S transition and cell proliferation. To examine whether the EFh domain is critical for the

role of IBA2 in regulating cell proliferation, we generated an IBA2 mutant in which the EFh

domain was deleted (Fig. 5E, left panel). A FLAG tag was added to the N terminus of the

deletion construct to monitor its expression by western blotting using an anti-FLAG antibody

(Fig. 5E, right panel). Deletion of the EFh domain reduced the effect of IBA2 in promoting

cell cycle progression (Fig. 5D), indicating that the EFh domain is essential for this function.

Transcriptional regulation of cyclin D1 by IBA2

Although cyclin D1 is overexpressed in ~50% of human breast cancers, cyclin D1 gene

amplification accounts for only 10% of these cases (31). Therefore, other mechanisms, such

as increased gene transcription, must be responsible for the overexpression of this cell cycle

regulator. To determine whether IBA2 regulates cyclin D1 expression in the transcription

level, we examined the effect of IBA2 on cyclin D1 promoter using luciferase reporter assays.

As shown in Fig. 6A, IBA2 induced expression of the cyclin D1 gene promoter in a




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dose-dependent manner in both MCF-7 cells and HEK 293 cells. These results indicate that

IBA2 upregulates cyclin D1 gene transcription.

We mapped cis-element(s) within the cyclin D1 promoter that is/are required for

IBA2-dependent gene transcription. MCF-7 cells were transfected with a series of

5’-truncated cyclin D1 promoter luciferase constructs (32) and with or without IBA2

expression constructs, and luciferase activity was measured (Fig. 6B). Deletion of the

sequence spanning -186 bp to -106 bp resulted in decrease in IBA2-dependent gene

transcription. Further deletion up to -53 bp led to a complete loss of IBA2-dependent gene

transcription. These results reveal that the cyclin D1 promoter region spanning nucleotides

-186 to -53 harbors essential element(s) responsible for mediating IBA2-dependent gene

transcription.

To determine if IBA2 directly regulates cyclin D1 gene transcription, we performed ChIP

assays to determine whether IBA2 is directly recruited to the cyclin D1 promoter. As shown in

Fig. 6C, IBA2 was recruited to the proximal region of the cyclin D1 promoter (-86 to -234 bp).

The recruitment of IBA2 was sequence-specific since negligible binding was detected either

with the genomic sequence 2.5 kb upstream of the cyclin D1 gene transcription start site or

with a control antiserum. This result demonstrates that IBA2 physically associates with the

cyclin D1 promoter.

To determine which domain of IBA2 is required for cyclin D1 gene transcription, we

tested the EFh domain deletion mutant of IBA2. Deletion of the EFh domain was associated

with a diminished IBA2-dependent cyclin D1 gene transcription (Fig. 6D), indicating that the

EFh domain of IBA2 is important for cyclin D1 gene transcription.




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IBA2 is overexpressed in human mammary tumors and its level is positively correlated

with tumor grade

Using immunohistochemistry (IHC), we screened paraffin-embedded mammary tissue

sections from 24 normal and 30 breast cancer patients for the expression of IBA2. While

normal mammary epithelial cells displayed no or weak IBA2 staining (Fig. 7A, left panel),

breast carcinoma cells displayed strong IBA2 staining in both the cytoplasm and nucleus (Fig.

7A, right panel).

To determine whether there is a correlation between the expression level of IBA2 and the

development and progression of breast tumors, we measured IBA2 mRNA expression levels

in primary tumors using quantitative real-time PCR. We collected normal mammary tissue

from 24 normal patients and breast carcinoma tissue from 30 breast cancer patients. IBA2 was

significantly upregulated in tumors and its expression correlated positively with histological

grades (Fig. 7B). Increased IBA2 mRNA expression was also evident in the majority of breast

carcinomas compared with adjacent normal tissues (Fig. 7C). Cyclin D1 mRNA levels in

carcinoma samples were also analyzed and were plotted against the levels of IBA2 mRNA

(Fig. 7D). Statistical analysis found a Pearson correlation coefficient of 0.5679 (P < 0.0001),

indicating a strong positive correlation between the expression of these two genes in breast

carcinomas. These data further support a role for IBA2 in cyclin D1 regulation and in breast

carcinogenesis. Collectively, these data suggest that overexpression of IBA2 may be a

frequent event in human breast cancer and promotes the initiation and progression of breast

cancer.

Discussion




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EFh domain-containing transcription factors play important roles in carcinogenesis. They

have structural similarity and affect a wide range of biological processes. The activities of

these proteins are determined in part by a distinct pattern of subcellular localization and tissue

specific expression. IBA1, an EF protein and homolog of IBA2, may contribute to the

progression of epithelial-mesenchymal transition (EMT) (33,34) and is overexpressed in

human mammary tumors and cervical cancer tissues (18,35). Overexpression of IBA1

promotes the proliferation of human breast cancer cells and facilitates tumor growth in female

nude mice (10,18). These results indicate that IBA1 may function as an oncogene. IBA2 has

similar overall structure to, but distinct expression patterns from IBA1, which indicates that

IBA2 may have other important roles in carcinogenesis. However, the potential role in

carcinogenesis and the cellular mechanism of IBA2 have not been well characterized. In the

current study, we showed that IBA2 promotes cell proliferation and tumorigenesis in breast

cancer cells via regulating cyclin D1.

EFh proteins regulate the expression of downstream target genes and play a role in

carcinogenesis; therefore, target identification is essential to understand the oncogenic

potential of these proteins. In this study, we identified the cyclin D1 gene as a downstream

target of IBA2 in breast cancer cells. Cyclin D1 is a critical regulator of the G0/G1 to S

transition in the cell cycle; thus, its identification as the downstream target of IBA2 is

consistent with the effect of IBA2 in promoting the G0/G1 to S transition. On the other hand,

cyclin D1 expression or transcriptional activities can be regulated by oncogenes, growth

factors, ion channels and G-protein-coupled receptors (36). We show here IBA2 can regulate

cyclin D1 expression and transcriptional activities.




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Cyclin D1 amplification and/or overexpression is one of the most prevalent alterations in

breast carcinomas, occurring in ~50% of cases (31,37-39). The oncogenic activity of cyclin

D1 in mammary tissue was demonstrated in mouse models wherein cyclin D1 overexpression

led to the development of mammary carcinoma (40), whereas cyclin D1 ablation resulted in

mice resistant to cancer induced by several oncogenes (41). In addition, cyclin D1-null mice

exhibited defective postnatal mammary development as revealed by a lack of proliferation of

alveolar epithelial cells in response to the sex steroid milieu of pregnancy (42,43). In fact,

cyclin D1 is a classical oncogene that is also amplified and/or overexpressed in a substantial

proportion of other human cancers including parathyroid adenoma, colon cancer, lymphoma,

melanoma, and prostate cancer (44).

Another approach to understanding the oncogenic potential of IBA2 is to decipher its

molecular actions, that is, the molecular details involved in its regulation of transcription.

IBA1 promotes human vascular smooth muscle cell proliferation (45,46) and this ability is

lost by mutations in the EFh region (47). IBA2 binds specifically to DNA sequences through

its EFh domain. EFh proteins activate multiple targets that are involved in cell growth, cell

cycle regulation, transcription and other cellular activities (1,2,48,49). As a result, IBA2

requires formation of stable protein-DNA complexes to promote cell proliferation and

tumorigenesis by regulating cyclin D1.

In summary, we report here that IBA2 is overexpressed in breast carcinomas. We have

demonstrated that IBA2 is able to promote cell proliferation and tumorigenesis of breast

cancer, possibly by facilitating the G1/S transition in the cell cycle. We identified the cyclin

D1 gene as a downstream target of IBA2 in breast cancer cells. These results will further our




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understanding of the oncogenic potential of IBA2, which will be useful for the prognosis and

therapy of breast cancer.

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Figure legends

Figure 1. Cloning and sequence analysis of IBA2. A, schematic diagram of IBA2 protein

sequence. The conserved EFh domain is shown. B, sequence analysis of IBA2 proteins from

different species. Boxed residues represent conserved regions. One potential phosphorylation

site (residue 2) is indicated with ▲, and one potential acetylation site (residue 33) is labeled

with ●. C, phylogenetic analysis of the evolutionary relationship among IBA2 family proteins

in different species.

Figure 2. The expression profile of IBA2 in mouse tissues and cell lines. A, real-time PCR

analysis of the tissue-specific IBA2 mRNA expression. B, western blotting analysis of IBA2

protein. MCF-7 cells were transfected with an empty vector or FLAG-IBA2. Cellular proteins

were prepared and western blotting was performed with anti-FLAG (left panel) or anti-IBA2

(right panel). C, real-time PCR (left panel) and western blotting (right panel) analysis show

that IBA2 is expressed at higher levels in breast cancer cells than in normal human breast

epithelial cells (MCF-10A). GAPDH and α-tubulin were used as internal controls. D,

subcellular localization of IBA2 protein. HeLa cells were transfected with EGFP-IBA2 (upper

panel) or FLAG-IBA2 (lower panel). After 24 h, EGFP fluorescence or immunofluorescence




18

(rhodamine) of the FLAG tag was visualized by fluorescence microscopy. DAPI staining was

included to visualize the cell nucleus.

Figure 3. IBA2 promotes proliferation of breast cancer cells. A, the effect of IBA2

overexpression on cell cycle progression. MCF-7 cells were transfected with an empty vector

or FLAG-IBA2 for 24 h and then switched to conditioned medium without serum for 24 h.

The cells were then cultured in medium containing 10% FBS for 24 h and collected for cell

cycle analysis by flow cytometry. The data shown in the right panel represent data from three

independent experiments. Representative profiles are shown in the left panel. B, the effect of

IBA2 knockdown on cell cycle progression. After MCF-7 cells were transfected with control

siRNA or IBA2 siRNA for 24 h, cells were switched to conditioned medium without serum

for 24 h. The cells were then cultured in medium containing 10% FBS for 24 h and collected

for cell cycle analysis by flow cytometry. The data shown in the right panel represent data

from three independent experiments. Representative profiles are shown in the left panel. C,

colony formation assay. MCF-7 cells stably expressing the indicated plasmids were

maintained in culture media for 14 days and stained with crystal violet. The number of

colonies in each condition was counted and expressed as the mean ± S.D. from triplicate

experiments. *p < 0.05 (two-tailed unpaired t-test). D, E, effect of IBA2 on cell proliferation.

MCF-7 cells (D) and HEK 293 cells (E) were transfected with the indicated constructs, and

cell proliferation was measured by BrdU incorporation. Data are means ± S.D. from triplicate

experiments. *p < 0.05 (two-tailed unpaired t-test).

Figure 4. IBA2 promotes tumorigenesis of breast cancer cells. A, B, C, IBA2 promotes breast

tumorigenesis. IBA2-overexpressing or IBA2 shRNA-expressing MCF-7 cells were




19

transplanted into ovariectomized athymic mice. Tumors were measured weekly using a

Vernier caliper and the volume was calculated according to the formula: π/6 × length × width2.

Representative images of tumor-bearing mice (A), the average tumor mass of each group (B),

and the growth curves of tumors (C) are shown. Each point represents the mean ± S.D. *p <

0.05 (two-tailed unpaired t-test).

Figure 5. Identification of cyclin D1 as a downstream target of IBA2. A, western blotting

analysis of the main proteins involved in the G1/S transition in IBA2 overexpressing or IBA2

knockdown cells. B, real-time PCR analysis of the expression of IBA2 and cyclin D1 in IBA2

overexpressing or IBA2 knockdown cells. C, the effect of cyclin D1 and IBA2 knockdown on

cell proliferation. D, the effect of IBA2-ΔEFh on cell proliferation. E, functional domain of

IBA2 implicated in the cell proliferative effect.

Figure 6. Transcriptional regulation of cyclin D1 by IBA2. A, IBA2 transactivates cyclin D1

promoter expression. MCF-7 (left panel) and HEK 293 cells (right panel) were co-transfected

with a cyclin D1-Luc reporter construct, a Renilla construct and different amounts of the

IBA2 expression construct (0.05, 0.2, and 0.8 µg/well). After 24 h, cells were harvested and

assayed for luciferase activity. B, mapping of cis-acting elements in the cyclin D1 promoter

that are responsible for IBA2 transactivation. Shown is a schematic representation of the

5’-truncated cyclin D1 promoter luciferase constructs and their activities in MCF-7 cells.

Cells were transiently transfected with the indicated reporter plasmids with or without

co-transfection of an IBA2 expression construct (0.8 µg/well). C, the recruitment of IBA2 to

the cyclin D1 promoter. Soluble chromatin from MCF-7 cells was immunoprecipitated with

anti-IBA2 or a control rabbit normal IgG. The final DNA extractions were amplified by PCR




20

and real-time PCR using primers that cover the proximal promoter region of the cyclin D1

gene or the upstream control region. D, functional domain of IBA2 implicated in its

transactivation activity of cyclin D1. Shown are the transactivation activities of the IBA2

deletion mutant on the cyclin D1 promoter. Data are means ± S.D. from triplicate

experiments.

Figure 7. IBA2 is overexpressed in human mammary tumors and its expression level

correlates with tumor grade. A, representative IHC of IBA2 protein in paraffin-embedded

human primary breast cancer (right panel) and adjacent normal tissue (left panel). H&E

staining shows standard morphology. B, IBA2 expression levels in normal and carcinoma

breast samples. Normalized IBA2 mRNA expression was measured by quantitative real-time

PCR using GAPDH mRNA as the internal control. C, IBA2 mRNA levels in paired samples

of breast carcinomas compared to adjacent normal mammary tissues. D, The relative level of

IBA2 expression was plotted against the relative level of cyclin D1 expression.




Figure 1

A

B

EFh

IBA2 150 aa

C

Nucleotide Substitutions (x100)

0

273.7

50100150200250

Homo sapiens

Mus musculus

Rattus norvegicus

Danio rerio

Gallus gallus

M S G E L S N R F Q G G K A F G L L K A R Q E R R L A E I N 1 Homo sapiens

M S V A L S N R F Q G G K A F G L L K A R Q E K R L E E I N 1 Mus musculus

M S V A L S N R F Q G G K A F G L L K A R Q E K R L E E I N 1 Rattus norvegicus

- - M P S N M D L Q G G K A F G L L K A Q Q R D K L E E I N 1 Danio rerio

M A A P R R P S G G G V R R A P Q D G R L E E I N K E F L C 1 Gallus gallus

R E F L C D Q K Y S D E E N L P E K L T A F K E K Y M E F D 31 Homo sapiens

R E F L C D Q K Y S D E E N L P E K L A A F K E K Y M E F D 31 Mus musculus

R E F L C D Q K Y S D E E N L P E K L A A F K E K Y M E F D 31 Rattus norvegicus

K E F M E D Q K Y R D E E D L P E K L D S F K N K Y A E F D 29 Danio rerio

D P K F S D E E D L E E K L A V F K E K Y M E F D L N N Q G 31 Gallus gallus

L N N E G E I D L M S L K R M M E K L G V P K T H L E M K K 61 Homo sapiens

L N N E G E I D L M S L K R M M E K L G V P K T H L E M K K 61 Mus musculus

L N N E G E I D L M S L K R M M E K L G V P K T H L E M K K 61 Rattus norvegicus

L N D Q G E I D M M G L K R M M E K L G V P K T H L Q M K K 59 Danio rerio

E I D L M S V K R M M E K M G V P K T H L E L K K M I S E V 61 Gallus gallus

M I S E V T G G V S D T I S Y R D F V N M M L G K R S A V L 91 Homo sapiens

M I S E V T G G V S D T I S Y R D F V N M M L G K R S A V L 91 Mus musculus

M I S E V T G G V S D T I S Y R D F V N M M L G K R S A V L 91 Rattus norvegicus

M I S E V T G G C S D T I N Y R D F V K M M L G K R S A V L 89 Danio rerio

T G G V S E T I S Y Q D F V N V M L G K R S A V L K L V M M 91 Gallus gallus

K L V M M F E G K A N E S S P K P V G P P P E R D I A S L P 121 Homo sapiens

K L V M M F E G K A N E S S P K P A G P P P E R D I A S L P 121 Mus musculus

K L V M M F E G K A N E S S P K P A G P P P E R D I A S L P 121 Rattus norvegicus

K L V M M F E D K A N G S S C K P D G P P P K R D I T S L P 119 Danio rerio

F E G K A N E S N P K P S G P P P E R D I A S L P 121 Gallus gallus

▲ ●




Figure 2

Re

lative

mR

NA

exp

ressio

n

IBA2

0

0.5

1

1.5

A

IBA2

WB: Anti-FLAG

kD

25

16.5 IBA2

WB: Anti-IBA2

B

D

DAPI GFP-IBA2 Merged

DAPI FLAG-IBA2 Merged

C

sp

leen

lun

g

live

r

ske

leta

l m

uscle

he

art

bra

in

kid

ney

te

stis

IBA2

G3PDH

kD

25

16.5

α-tubulin




0

40

80

120G1 S G2/M

0

40

80

120G1 S G2/M

0

0.5

1

1.5

0

0.5

1

1.5

0

0.5

1

1.5

0

0.5

1

1.5

0

0.5

1

1.5

0

0.5

1

1.5

Figure 3

C

D

*

*

Rela

tive c

olo

ny n

um

be

r

*

*

Vector IBA2

Perc

enta

ge

Control IBA2 RNAi

Perc

enta

ge

Rela

tive B

rdU

incorp

ora

tion

Vector IBA2

Control IBA2 RNAi

Vector IBA2

Vector IBA2

Control IBA2 RNAi

Rela

tive c

olo

ny n

um

be

r

MCF-7 HEK 293

*

*

Vector IBA2 Control IBA2

RNAi

E

Rela

tive B

rdU

incorp

ora

tion

Control IBA2

RNAi

A

G0/G1: 69.14 %

G2/M: 5.60 %

S: 25.26 %

G0/G1: 55.79 %

G2/M: 12.72 %

S: 31.48 %

Vector IBA2

G0/G1: 62.39 %

G2/M: 12.44 %

S: 25.17 %

G0/G1: 69.51 %

G2/M: 13.68 %

S: 16.81 %

Control IBA2 RNAi

Num

ber

0

400

800 1

200

0 40 80 120

Channels (FL2-A)

Num

ber

0

200

400

600

800

0 40 80 120

Channels (FL2-A)

Num

ber

0

200

400

600

0 40 80 120

Channels (FL2-A)

Num

ber

0

400

800 1

200

0 40 80 120

Channels (FL2-A)

B




0

0.5

1

1.5

2

Figure 4

Tu

mo

r vo

lum

e (

mm

3)

A

C

Weeks

We

igh

t (g

)

0

400

800

1200

1600

2000

1 2 3 4 5 6 7 8

Vector

IBA2

Control

IBA2 RNAi

B

* *




0

0.5

1

1.5IBA2 CyclinD1

0

4

8

12

16IBA2 CyclinD1

IBA2

A B

Vector IBA2 CDK2

CDK4

P-Rb (Ser-780)

Figure 5

Re

lative

mR

NA

le

ve

l R

ela

tive

mR

NA

le

ve

l

Cyclin B1

CDK6

P21

P53

Cyclin D3

Cyclin E1

Cyclin D1

0

40

80

120G1 S G2/M

D

Vector IBA2 IBA2 (DEFh)

C

Control IBA2 RNAi IBA2 RNAi

+Cyclin D1

WT

Del

EFh

WB：IBA2

E

Pe

rcenta

ge

Control IBA2 RNAi α-tubulin

0

40

80

120G1 S G2/M

Pe

rcenta

ge




0

1

2

3

4

5

0

5

10

NegtiveControl

CyclinD1

IgG

IBA2

Input IgG IBA2

A

C

CyclinD1

D

Figure 6

Cyclin D1-Luc

IBA2

Re

lative

lu

cife

rase

activity

Negtive Control

0

5

10

15

0

5

10

B

0 1 2 3 4 5 6 7

SOX2

Vector

-962

Luciferase

-605

-53

-186

-106

-844

Luciferase

Luciferase

Luciferase

Luciferase

Luciferase

Luciferase

Luciferase

0

-1745

Relative luciferase activity

0 1 2 3 4 5 6 7

SOX2

Vector

-962

LuciferaseLuciferase

-605

-53

-186

-106

-844

Luciferase







0

-1745


IBA2

Vector

LUC

-1745

-962

-844

-605

-186

-106

-53

0

LUC

LUC

LUC

LUC

LUC

LUC

LUC

MCF-7 HEK 293

IBA2 - - -

IBA2 (DEFh) - - -

Cyclin D1-Luc

Fo

ld e

nrichm

ent


Re

lative

lu

cife

rase

activity




0

4

8

12

Figure 7

Number 24 15 15

Mean 1.028 2.346 4.126

S.D. 0.705 1.920 3.647

Normal Grade II Grade III Re

lative

mR

NA

le

ve

l o

f IB

A2

B A

Normal Carcinoma

Re

lative

mR

NA

le

ve

l

Sample pairs

C

IHC

HE

* **

0

2

4

6

8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Adjacent Tumor

***

***

***

***

***

***

**

**

***

***

* *

0

2

4

6

8

0 2 4 6 8 10Re

lative

mR

NA

le

ve

l o

f C

yclin

D1

Relative mRNA level of IBA2

D




Published OnlineFirst June 4, 2016.Cancer Res Ying Zhang, Shuling Wang and Lingsong Li cancers via transcriptional control of cyclin D1EF hand protein IBA2 promotes cell proliferation in breast

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